Fl 393

H1299 cells (p53 null) were co-transfected with FLAG-HA-EZH2 and WT or mutant p53, respectively, or transfected with FLAG-HA-EZH2 alone. Nuclear extract (NE) was prepared from these cells and incubated with a polyclonal p53 antibody (FL393, Santa Cruz) prior to addition of protein G beads. After overnight incubation, beads were then washed five times and eluted with glycine (0.1 M, pH 2.0), and then neutralized by adding Tris solution (1.5 M, pH 8.8). The eluates were mixed with SDS sample buffer and analyzed by SDS-PAGE, followed by immunoblotting^{58 (link)}.

Cells or tissues were lysed with LB (125 mM Tris-HCl pH 6.7, NaCl 150 mM, NP40 0.5%, SDS 0.1%, 10% Glycerol). Proteins were denatured and deposited directly (40 μg of proteins) onto SDS PAGE gels. Western blotting was performed using antibodies raised against p53 (rabbit anti-p53, FL-393, Santa Cruz, 10410 Finnell Street, Dallas, TX 75220, USA), p73 (rabbit anti-p73, Ab40658, Abcam, 24 rue Louis Blanc, 75010 Paris, France), HDAC4 (rabbit anti-HDAC4, 607702, Biolegend, France 38 Rue de Berri, 75008 Paris 8, France), acetylated Tubulin K40 (Merck, 201, Rue Carnot Fontenay sous BoisÎle-de-France 94126, France), and cleaved caspase 3 (cCASP3, #9661 Cell Signaling, Cell Signaling Technology Dellaertweg 9b 2316 WZ Leiden, Netherlands). Secondary antibodies (antirabbit NA934V and antimouse NXA931V Horseradish linked) were incubated at 1:10,000. Loading was controlled by analyzing actin protein expression (mouse anti-actin Clone C4, Chemicon, 1:10.000) [52 (link)]. Western blot quantifications were performed using the Pxi imager and Genetools (Syngene^TM, Beacon House Nuffield Road Cambridge CB4 1TF, United Kingdom). The intensity of the bands is indicated as a percentage (%) relative to control when a band is present in the control condition. If not, intensity is indicated as relative to a selected condition as indicated in the figure legends.

U87MG were treated with DMSO (control), EB148 or EB54 for 16 h. When indicated, after 16 h of incubation, cells were washed twice with fresh saline, and incubated with fresh medium not containing the drugs for an additional 24 h. At the end of the treatment period, the cells were collected and then lysed for 60 min at 4°C using 200 μl of RIPA buffer (9.1 mM NaH₂PO₄, 1.7 mM Na₂HPO₄, 150 mM NaCl, pH 7.4, 0.5% sodium deoxycholate, 1% Nonidet P-40, 0.1% SDS, and a protease-inhibitor cocktail). Equal amounts of the cell extracts (40 μg of protein) were diluted in Laemmli sample solution, resolved using SDS-PAGE (8.5%), transferred to PVDF membranes and probed overnight at 4°C using an anti-p53 antibody (FL-393, Santa Cruz Biotechnology; 1:500). Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was the loading control (G9545, Sigma Aldrich, Milan, Italy, 1:5000). The primary antibodies were detected using the appropriate peroxidase-conjugated secondary antibodies, which were then detected using a chemioluminescent substrate (ECL, Perkin Elmer). Densitometric analysis of the immunoreactive bands was performed using Image J Software.